MicroRNA-Mediated Downregulation of HMGB2 Contributes to Cellular Senescence in Microvascular Endothelial Cells

High mobility group box 2 (HMGB2) is a non-histone chromosomal protein involved in various biological processes, including cellular senescence. However, its role in cellular senescence has not been evaluated extensively. To determine the regulatory role and mechanism of HMGB2 in cellular senescence, we performed gene expression analysis, senescence staining, and tube formation assays using young and senescent microvascular endothelial cells (MVECs) after small RNA treatment or HMGB2 overexpression. HMGB2 expression decreased with age and was regulated at the transcriptional level. siRNA-mediated downregulation inhibited cell proliferation and accelerated cellular senescence. In contrast, ectopic overexpression delayed senescence and maintained relatively higher tube-forming activity. To determine the HMGB2 downregulation mechanism, we screened miRNAs that were significantly upregulated in senescent MVECs and selected HMGB2-targeting miRNAs. Six miRNAs, miR-23a-3p, 23b-3p, -181a-5p, -181b-5p, -221-3p, and -222-3p, were overexpressed in senescent MVECs. Ectopic introduction of miR-23a-3p, -23b-3p, -181a-5p, -181b-5p, and -221-3p, with the exception of miR-222-3p, led to the downregulation of HMGB2, upregulation of senescence-associated markers, and decreased tube formation activity. Inhibition of miR-23a-3p, -181a-5p, -181b-5p, and -221-3p delayed cellular senescence. Restoration of HMGB2 expression using miRNA inhibitors represents a potential strategy to overcome the detrimental effects of cellular senescence in endothelial cells.


Introduction
In 1965, Hayflick et al. first used the term "cellular senescence" to describe the limitations of normal human cell proliferation [1]. Cellular senescence is characterized by a stable and irreversible cell cycle arrest, and is associated with multiple cellular and molecular changes. Cellular senescence can compromise tissue repair and regeneration, thereby contributing to aging. Removal of senescent cells can attenuate age-related tissue dysfunction and extend health span [2]. The causes of cellular senescence involve telomere shortening, DNA damage, oxidative stress, and oncogene activation [3]. Telomere shortening generated by repeated DNA replication is mainly responsible for replicative senescence [2,4]. DNA damage induced by radiation and reactive oxygen species resulting in double-stranded DNA break is a potent inducer of stress-induced premature senescence [5,6]. Activation of oncogenes or inactivation of tumor suppressors is a major cause of oncogene-induced senescence [2,6,7]. Activation of the p53/p21 WAF1/CIP1 and p16 INK4A /pRB tumor suppressor pathways play a central role in regulating senescence [8].
The vascular endothelial structure consists of endothelial monolayer cells that can exchange substances, such as gases and nutrients, between cells and blood. Endothelial cells represent an important component of this function, and the lack or dysfunction of these cells in the vascular system damages the circulatory system [18]. Cellular senescence in endothelial cells results in endothelial barrier dysfunction and plays an important role in the risk of various cardiovascular diseases [19]. Endothelial function is modulated by traditional cerebrovascular disease risk factors in young adults; however, aging is independently associated with the development of vascular endothelial dysfunction [27,28].
High mobility group box 2 (HMGB2) proteins are the most abundant non-histone chromatin-binding proteins in the nuclei of mammalian cells. It has been proposed that when HMGB proteins bind to chromatin, they locally modify the structure by bending DNA and facilitating the binding of regulatory proteins such as transcription factors, chromatin remodelers, and DNA damage repair machinery [29][30][31]. HMGB proteins control several genomic processes in response to specific biological cues via their interaction with chromatin; therefore, they represent essential regulators of cellular programs as well as disease [32][33][34][35][36]. Previously, we observed that HMGB2 is downregulated by p21 during radiation-induced senescence via the ATM-p53-p21 DNA damage signaling cascade [37]. Therefore, we examined whether HMGB2 is also involved in replicative senescence. In this study, we found that HMGB2 expression decreased with aging, and this regulation was mediated via SA-miRNAs which target HMGB2. Restoration of HMGB2 by targeting these miRNAs represents a potential strategy to overcome the detrimental effects of cellular senescence in endothelial cells.

Cell Culture
Human lung microvascular endothelial cells (MVECs) were purchased from Cell Applications (San Diego, CA, USA) and cultured in EBM-2 media supplemented with EGM-2MV (Lonza, Hopkinton, MA, USA). In total, 5 × 10 5 cells were plated in a 100 mm culture plate and cultured at 37 • C in a humidified incubator with 5% CO 2 . The cells were passaged every 3 d via trypsinization. Cell counts were determined at the end of every passage after staining with 0.1% trypan blue using a hemocytometer. The cumulative number of population doublings (PDLs) was calculated in relation to the initial cell number.

Telomere Length Assay
Telomere length measurement was performed by quantitative polymerase chain reaction as per the standard procedure 21369534 [38]. Telomere standard curve was generated by plotting threshold cycle (Ct) values against the amount of telomere sequence in kb per reaction suing a ten-fold serial dilution of a synthetic oligonucleotide. Genomic DNA was extracted from MVECs using GeneAll Exgene Cell SV mini kit (GeneAll, Seoul, Korea). Amplification was performed on a Bio-Rad CFX96 machine (Bio-Rad Laboratories, Hercules, CA, USA) in a final volume of 20 µL containing 1× KAPA SYBR ® FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA), 100 nM of both pairs of primers, and 20 ng of genomic DNA. Cycling condition was as follows: 3 min at 95 • C followed by 35 cycles at 98 • C for 7 s and 60 • C for 20 s, followed by a melt curve. Single-copy gene, 36B4, served as a reference gene to normalize the Ct values from the telomere assay. Amplification was performed with 1 µg of genomic DNA as above and cycling condition was as follows: 3 min at 95 • C followed by 35 cycles at 98 • C for 7 s and 58 • C for 20 s. Absolute telomere length was calculated and the results are expressed in kb/genome.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
For the analysis of mRNA expression, total RNA was isolated using a Hybrid-R Total RNA Purification kit (GeneAll). cDNA was synthesized using the PrimeScript RT Master Mix kit (TaKaRa Bio, Kusatsu, Japan), and quantitative PCR was performed using the qPCR Green 2X master mix (MBiotech, Seoul, Korea) on a Bio-Rad CFX96 machine (Bio-Rad Laboratories). Gene expression was normalized to that of two reference genes (PPIA and RPL13A), and the relative gene expression values were calculated based on the Ct value using the 2-∆∆Ct method [32]. For the analysis of miRNA expression, total RNA was isolated using the TRIzol reagent (Thermo Fisher Scientific) and cDNA was synthesized using the Mir-X™ miRNA First-Strand Synthesis Kit (Clontech Laboratories, Palo Alto, CA, USA). Quantitative PCR was performed using the qPCR Probe 2× Master Mix (MBiotech, Seoul, Korea). The primers used for qRT-PCR are listed in Supplementary Table S1.

Senescence-Associated β-Galactosidase (SA-β-gal) Staining
Cellular SA-β-gal activity was measured as described previously [33]. Briefly, 1.5 × 10 5 cells were plated in a 60 mm dish and cultured for 3 d. Cells were washed with phosphate-buffered saline (PBS) and fixed with 2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in PBS for 10 min at room temperature. The presence of SA-β-gal activity was determined by incubating the cells in a solution containing 40 mM citric acidsodium phosphate (pH 6.0), 150 mM NaCl, 2 mM MgCl 2 , 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml X-gal for 14 h at 37 • C in a dark incubator. Cells were counterstained with eosin solution (100 mg eosin Y, 0.5% (v/v) acetic acid in 80% ethyl alcohol) and the proportion of blue cells observed under a light microscope was measured.

Tube Formation Assay
Tube formation was evaluated as described previously [34,35]. Briefly, 5 × 10 4 MVECs were seeded onto each well of 24-well plates pre-coated with BD Matrigel Matrix (BD Biosciences, San Jose, NJ, USA) and incubated overnight to allow the formation of tubelike structures. Endothelial cell tube formation was assessed using a GE InCell Analyzer 2000 (GE Healthcare Life Sciences, Little Chalfont, UK) and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA) using the Angiogenesis Analyzer plugin.

Transfection of Small Interfering RNA (siRNA), miRNA, and miRNA Inhibitors
The siRNAs and synthetic miRNA mimics used in this study were synthesized by Genolution Pharmaceuticals (Seoul, Korea). miRNA inhibitors involving 2 -O-methylmodified oligoribonucleotide single strands were purchased from Genolution Pharmaceuticals and Integrated DNA Technologies (Singapore). Briefly, 1.5 × 10 5 cells were reverse-transfected with 20 nM siRNA/miRNA or 40 nM miRNA inhibitor using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) in a 60 mm dish, according to the manufacturer's instructions. The sequences of the siRNA and miRNA mimics are listed in Supplementary Table S2.

Small RNA Sequencing
Total RNA was extracted from young (PDL23) and old (PDL49) MVECs using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality check, library preparation, next generation sequencing, and data analysis were performed by ebiogen (Seoul, Korea), and the small RNA sequencing data were deposited in the GEO database (GSE192677).

Statistical Analysis
Data are presented as the mean ± standard error. All results were analyzed for statistical significance using one-way and two-way analysis of variance (ANOVA) with Fisher's PLSD post-hoc test. A p-value of 0.01 (**) and 0.001 (***) was compared with that of the control group. Statistical analyses were performed using StatView version 5.0.1.

HMGB2 Expression Decreased in Aged MVECs
To determine the role of HMGB2 in the aging of microvessels and aging-related vascular disease, we used primary MVEC cultures as a replicative senescence model. Replicative senescence was induced by continuously subculturing early passaged MVECs at PDL20 until they reached the end of their lifespan (PDL51) ( Figure 1A). The absolute telomere length (aTL) was also measured to demonstrate that senescence was indeed triggered by telomere shortening. As cell proliferation was found to slow down from PDL42 onwards, we considered that cellular senescence was initiated from this point for convenience. Young (PDL28), early senescent (PDL45), and fully senescent (PDL51) MVECs were subjected to SA-β-Gal histochemical staining ( Figure 1B). Senescent MVECs exhibited poor growth and senescent morphology with strong SA-β-Gal staining. Under the same conditions, we measured HMGB2 protein levels using western blot analysis ( Figure 1C). Similar to other types of cells, HMGB2 levels were decreased and the expression of two canonical senescence markers, p16 and p21, was induced during cellular senescence. To verify whether this decrease in HMGB2 level during cellular senescence is regulated at the transcriptional level, qRT-PCR was performed. HMGB2 mRNA levels gradually decreased with the population doubling, showing an inverse correlation with the transcription of p16 and p21 genes ( Figure 1D). The mRNA expression levels of HMGB2, p16, and p21 were quantified via quantitative real timepolymerase chain reaction. HMGB2, high mobility group box 2; MVECs, microvascular endothelial cells; PDL, population doubling; aTL, absolute telomere length; SA-β-gal, senescence-associated beta galactosidase. *** p < 0.001.

HMGB2 Silencing Induced Premature Senescence of MVECs
To determine the effect of HMGB2 downregulation on cellular senescence, HMGB2 was downregulated in young MVECs (PDL28) via treatment with siRNA which targeted HMGB2. After transfection, HMGB2 knockdown was confirmed via western blot analysis (Figure 2A). HMGB2 knockdown was associated with classical features of cellular senescence in young MVECs, as demonstrated by an impaired cell proliferative potential ( Figure 2B) and an increased proportion of SA-β-gal-positive cells ( Figure 2C). Tube formation assay is one of the most widely used in vitro assays to model the reorganization of angiogenesis, and measures the ability of endothelial cells to form capillary-like structures. HMGB2 knockdown caused MVECs to form a tube structure in a less efficient manner than that observed in the control ( Figure 2D). These results suggest that HMGB2 depletion contributes to premature senescence in MVECs. The effect of HMGB2 knockdown on the angiogenic activity of MVECs was analyzed via in vitro tube formation assay and quantified using Image J software with the Angiogenesis Analyzer plugin. siCTR, small interfering RNA control; siHMGB2, small interfering RNA against HMGB2. ** p < 0.01, *** p < 0.001.

HMGB2 Overexpression Delayed Replicative Senescence in MVECs
Next, we investigated whether ectopic overexpression of HMGB2 can delay senescence progression. Retrovirus-mediated HMGB2 overexpression was established in exponentially growing MVECs (PDL30) and serially passaged cells. HMGB2 overexpression was maintained in serially passaged cells (PDLs 36-45) ( Figure 3A). Overexpression of HMGB2 did not substantially increase the proliferative potential of cells compared to that in the vectoronly control ( Figure 3B), although it could not rejuvenate MVECs ( Figure 2B). In addition, HMGB2 overexpression decreased the proportion of SA-β-gal-positive cells ( Figure 3C) and maintained relatively better tube formation activity ( Figure 3D). Therefore, ectopic overexpression of HMGB2 may help MVECs maintain a healthy state.

Screening of SA-miRNAs That Target HMGB2 in MVECs
Growing evidence suggests that miRNAs act as inducers of senescence [40,41]. Therefore, we speculated that miRNAs targeting HMGB2 may be induced which downregulate HMGB2, leading to senescence in MVECs. As senescent cells of different lineages demonstrate tissue-specificity in miRNA profiles [42], we performed small RNA sequencing to screen for miRNAs specifically induced in senescent MVECs (PDL49) relative to proliferating cells (PDL23). miRNA candidates that targeted HMGB2 were searched based on the 3 UTR sequence of HMGB2 using seven sequence-based miRNA search platforms (Supplementary Table S3). All miRNA candidates were pooled (n = 75), and we selected miRNAs recommended by more than three platforms (n = 25) (Supplementary Table S4). Among them, six miRNAs with sufficient read numbers were selected for further analysis ( Figure 4A). The sequence alignment of miRNAs with the possible target sequences in the 3 UTR of HMGB2 are shown in Figure 4B. The expression of selected miRNAs in serially passaged MVECs was analyzed via qRT-PCR, which confirmed that all six miRNAs were induced in senescent MVECs ( Figure 4C).

HMGB2-Tageting SA-miRNAs Regulated Cellular Senescence in MVECs
To identify miRNAs that downregulate HMGB2 in vivo, we transfected the synthetic miRNA mimics into MVECs and performed western blotting ( Figure 5A). Among the six candidates, all candidates except miR-222-3p downregulated HMGB2, which confirmed that the HMGB2 gene is the target of these miRNAs in MVECs. Next, we confirmed the target specificity of miRNAs using the luciferase reporter system since miR-221-3p and miR-222-3p share a common target sequence ( Figure 4B). All miRNAs except for miR-222-3p repressed luciferase activity ( Figure 5B) when the luciferase reporter system containing the 3 UTR sequence of HMGB2 was co-transfected with miRNA. We then confirmed that the introduction of exogenous miRNA accelerated cellular senescence, and the proportion of SA-β-gal-positive cells increased ( Figure 5C). In addition, five miRNA candidates inhibited angiogenic tube formation ( Figure 5D). These results suggest that miR-23a-3p, miR-23b-3p, miR-181a-5p, miR-181b-5p, and miR-221-3p represent SA-miRNAs that target HMGB2 in MVECs.

Inhibition of HMGB2-Targetting SA-miRNAs Delayed Senescence in MVECs
To confirm the effects of the selected miRNAs on cellular senescence, we used a miRNA inhibitor, a steric-blocking oligonucleotide that hybridizes to mature miRNAs and inhibits their function. Transfection of miRNA inhibitors into MVECs (PDL42) was associated with relatively higher expression levels of HMGB2 protein in senescent MVECs ( Figure 6A), which may be due to the prevention of HMGB2 downregulation induced by miRNA. As expected, these MVECs displayed a lower level of cellular senescence, a lower proportion of SA-β-gal-positive cells ( Figure 6B), and higher angiogenic tube formation activity ( Figure 6C) than those observed in cells treated with miRNA alone. These results suggest that miRNA-mediated downregulation of HMGB2 contributes to cellular senescence, and targeting miRNAs can delay senescence progression.

Discussion
HMGB2 is the most abundant non-histone chromatin-binding protein present in the nuclei of mammalian cells [43]. HMGB proteins are known to modulate the local chromatin environment, facilitating the binding of other proteins to chromatin, and controlling nuclear processes such as transcription, DNA damage repair, and nucleosome sliding [44]. Growing evidence supports the key role of HMGB2 in cellular senescence and related diseases. Aging-related loss of HMGB2 in articular cartilage is linked to reduced cellularity, which contributes to the development of osteoarthritis [45]. HMGB2 is involved in cell cycle arrest and chromatin remodeling during senescence. For example, HMGB2 binds to the senescence-associated secretory phenotype gene loci and protects loci from gene silencing via heterochromatin spreading [46]. A recent study has shown that HMGB2 is depleted from the nucleus upon initiation of cellular senescence, resulting in the reorganization of the genome and changes in transcriptional activity. HMGB2 modulates the global chromatin structure and expression of genes found within topologically associating domains by insulating against the clustering of CTCF proteins [47]. These studies support the crucial role of HMGB2 in senescence and aging processes. However, the mechanism of HMGB2 downregulation upon initiation of senescence has not yet been addressed. Studying the downregulation mechanism is important for controlling senescence and preventing the detrimental effects of senescence. In the present study, we showed that age-associated induction of miRNAs targeting HMGB2 contributes to the depletion of HMGB2 during replicative senescence in MVECs.
The overall HMGB2 level decreased with age ( Figure 1C), mostly at the transcriptional level ( Figure 1D). Although p21 inhibits HMGB2 transcription during radiation-induced senescence [37], p21 does not seem to be crucial in replicative senescence since HMGB2 depletion is followed by induction of p21. This signifies that the canonical DNA damage signaling, mediated by the ATM-p53 signaling pathway, does not significantly contribute to the downregulation of HMGB2, even if telomere erosion and subsequent DNA damage are important triggering events in replicative senescence. However, two canonical CDK inhibitors, p16 and p21, may play a role in accelerating the depletion of HMGB2 via cell cycle regulation. As E2F1 controls the timely expression of the S phase of the cell cyclespecific genes, including HMGB2 [48], activation of these CDK inhibitors and cell cycle arrest at the G1 phase inevitably leads to a decrease in HMGB2 transcription. Therefore, we speculate that the inhibition of HMGB2 transcription could have been an accompanying result of cell cycle arrest, and other mechanisms downregulating HMGB2 may function upon initiation of senescence.
In this study, we identified five SA-miRNAs targeting HMGB2 in MVECs: miR-23a-3p, miR-23b-3p, miR-181a-5p, miR-181b-5p, and miR-221-3p ( Figure 5). Although these miR-NAs have been reported to regulate senescence in other types of cells or angiogenesis in HUVEC, HMGB2 has not been considered a prime target of these miRNAs. While miR-23a-3p is reported to regulate dermal aging and senescence by targeting hyaluronan synthase 2 (HAS2) [49], miR-181a/b-5p contributes to the induction of senescence in primary human keratinocytes by targeting sirtuin 1 (SIRT1) [50]. Markopoulos et al. presented a distinct set of 15 miRNAs, including these five miRNAs, that are significantly upregulated in senescent human lung fibroblasts [51]. Based on pathway analysis of miRNA target genes, this subset of miRNAs acts in concert to induce cell cycle phase arrest and telomere erosion, establishing a senescent phenotype. In addition, miR-181a/b and miR-221 have been reported to modulate angiogenesis by targeting platelet-derived growth factor receptor A (PDGFRA) [52], and tissue inhibitor of metalloproteinase 3 (TIMP3) and hypoxia-inducible factor 1-α (HIF1A) [53,54], respectively. miR-222-3p inhibits migration and tube formation in endothelial progenitor cells by targeting adiponectin receptor 1 (ADIPOR1) [55]. Fibroblasts and endothelial cells have a common subset of miRNAs that can regulate senescence, even though specific and distinct expression of miRNAs has been reported.
Senescent cells accumulate in tissues and cause age-related decline and disease with aging. Studies show that there is a relationship between HMGB2 and cardiovascular diseases in cardiac myocytes [64,65]. Senescence in tissues is affected by the interaction with neighboring cells in the local microenvironment [66]. The findings of the present study can help prevent cardiovascular diseases and other related diseases. Restoring HMGB2 activity by using miRNA inhibitors can overcome the detrimental effects of aging and pathological remodeling.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/cells11030584/s1, Table S1: Primer sequences used for qRT-PCR, Table S2: Sequences of siRNA and miRNA mimics, Table S3: miRNA target prediction platform used in this study; Table S4: List of differentially expressed miRNAs targeting HMGB2.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.